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View additional product information for HEP50 Pooled Human Cryopreserved Hepatocytes - FAQs (HMCS50)
18 product FAQs found
Thermo Fisher Scientific has a dedicated team to answer questions regarding hepatocytes. You can send a question by email (hepaticproducts@thermofisher.com) or contact us by phone: US toll-free: (866) 952 3559
Our hepatocytes are prequalified and sold according to application, such as induction, short-term metabolism, and transporter uptake. If you require hepatocytes with particular P450 values or donor specifications, contact our Hepatic Biology Tech Support Team (hepaticproducts@thermofisher.com.com) or (866) 952-3559 to help with specific lot recommendations.
Thermo Fisher Scientific has a dedicated team to answer questions regarding hepatocyte cell culture. You can send a question by email (hepaticproducts@thermofisher.com) or contact us by phone: US toll-free: (866) 952-3559
Thermofisher Scientific works with a variety of human tissue sources, including Tissue and Organ Procurement Organizations, qualified Research Tissue Organizations and prominent academic and medical centers through collaborations that follow rigorous regulations, certifications and/or accreditations (“Source Facilities”). Tissues obtained through source facilities are consistent with the applicable legal and ethical practices of the United States.
Source Facilities are required by federal law to maintain accurate records and donor confidentiality as well as adhere to certain applicable laws governing tissue used for research globally. These include, but are not limited to:
- 45 CFR Part 46, subparts A, B, C, D where applicable (US federal guidance on the use of human subjects in research)
- Health Insurance Portability and Accountability Act (HIPAA) (addressing issues of confidentiality of personal information)
- National Organ Transplant Act (42 CFR Part 482)
- The Uniform Anatomical Gift Act (UAGA) of 1968, revised 1984
The human liver cells are derived from tissue obtained from accredited institutions. Consent is obtained by these institutions from the donor or the donor’s legal next of kin, for the use of the tissue and its derivatives for research purpose.
This could be due to the toxicity of the test compound. Here are other potential causes and recommendations:
-Sub-optimal culture medium: Use Williams Medium E with Plating and Incubation Supplement Packs; refer to our plating protocol
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating
-Cells were cultured for too long: In general, plateable cryopreserved hepatocytes should not be cultured for more than five days
First of all, we recommend comparing results to those reported on our lot-specific characterization specification sheet (human cells) and also referring to our enzyme induction protocol. Here are other potential causes and recommendations:
-Sub-optimal monolayer confluency: Please see our recommendations for 'I have a sub-optimal monolayer confluency for my hepatocytes. What should I do?'
-Poor monolayer integrity: Please see our recommendations for 'With my hepatocytes, I’m seeing rounding up of the cells, cellular debris, and/or holes in the monolayer, indicating dying cells. What should I do?'
-Inappropriate positive control: Check positive control to ensure suitability
-Incorrect concentration of positive control: Use the correct concentration of positive control
Please see the following causes and recommendations:
-Hepatocyte lot not transporter-qualified: Check lot specifications to ensure it is transporter-qualified
-Sub-optimal culture medium: Use Williams Medium E with Plating and Incubation Supplement Packs; refer to our plating protocol
-Not enough time for bile canaliculi to form: In general, at least 4-5 days in culture is required for bile canalicular network formation
Please see the following causes and recommendations:
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating
-Sub-optimal culture medium: Use Williams Medium E with Plating and Incubation Supplement Packs; refer to our plating protocol
-Cells were cultured for too long: In general, plateable cryopreserved hepatocytes should not be cultured for more than five days
Please see the following causes and recommendations:
-Seeding density too high: Check lot-specific characterization specification sheet for appropriate seeding density (human cells); observe cells under microscope for appropriate seeding prior to incubation
-Insufficient dispersion of hepatocytes during plating: Disperse cells evenly by moving plate slowly in a figure-eight and back-and-forth pattern in incubator; shake plate and wash cell monolayers prior to applying Geltrex Matrix overlay
-Improper plating volume used for well format : Refer to literature or technical support for suggested plating volumes
Please see the following causes and recommendations:
-Seeding density too low: Check lot-specific characterization specification sheet for appropriate seeding density (human cells); observe cells under microscope for appropriate seeding prior to incubation
-Insufficient dispersion of hepatocytes during plating: Disperse cells evenly by moving plate slowly in a figure-eight and back and forth pattern in incubator
-Insufficient plating volume used for well format: Refer to literature or technical support for suggested plating volumes
-Low attachment efficiency: Please see our recommendations for: 'I'm getting low attachment efficiency with my hepatocytes. What should I do?'
-Some animal lots are not greater than 80% confluent: Check lot-specific characterization specification sheet for appropriate seeding density. Note: Some animal species create chains or islands of cells rather than being 100% confluent.
Please see the following causes and recommendations:
-Not enough time for cells to attach: Wait before overlaying with Geltrex Matrix to see if attachment increases; compare cultures to pictures on the lot-specific characterization specification sheet (human cells)
-Poor-quality substratum: Use Gibco Collagen I-Coated Plates
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating; review thawing, plating, and counting protocols
Please see the following causes and recommendations:
-Improper thawing technique: Review thawing, plating and counting protocols; thaw cells <2 mins at 37 degrees C
-Sub-optimal thawing medium: Use HTM Medium during thawing to remove cryoprotectant
-Incorrect centrifugation speed: Check thawing protocol for proper centrifugation speed and time (varies by species; human is 100 x g for 10 min at RT)
-Rough handling of hepatocytes during counting: Mix slowly, use wide-bore pipette tips; ensure a homogenous cell mixture prior to counting
-Improper counting technique: Count cells on 2 of the 4 grid lines; do not let cells sit in trypan blue mixture for more than 1 min prior to loading
Please see the following causes and recommendations:
-Improper thawing technigue: Review thawing, plating and counting protocols; thaw cells less than 2 mins at 37 degrees C
-Sub-optimal thawing medium: Use HTM Medium during thawing to remove cryoprotectant
-Rough handling of hepatocytes during counting: Mix slowly, use wide-bore pipette tips; ensure a homogenous cell mixture prior to counting
-Improper counting technique: Count cells on 2 of the 4 grid lines; do not let cells sit in trypan blue mixture for more than 1 min prior to loading
-Cells left out too long: Plate cells immediately after counting
Unlike immortalized cell lines, hepatocytes are primary cells that cannot be cultured indefinitely. The use of thawed suspension hepatocytes should be limited to short-term experiments with a maximum of 4-6 hour incubations. Plateable hepatocytes, which attach to collagen-coated plasticware in culture media, are generally metabolically active for anywhere from 2-7 days depending on which assay they are qualified for use with.
If properly stored, cryopreserved hepatocytes can be maintained in the vapor phase of liquid nitrogen (-135 degrees C or below) for several years. This makes them ideal for a series of experiments conducted over many months.
Cryopreserved hepatocytes are shipped in the vapor phase of liquid nitrogen contained in a non-hazardous container called a dry liquid nitrogen (LN2) vapor shipper or dewar. The internal temperature of the dewar is maintained between -140 degrees C and -160 degrees C, generally for up to 7-10 days if unopened. Once hepatocytes are received and transferred to a long term LN2 dewar in the laboratory, dewers are returned to the address listed on the pre-paid return shipping label for reuse. Upon receipt of a shipment of cryopreserved hepatocytes, carefully and quickly transfer the vials to the vapor phase of liquid nitrogen and keep at -135 degrees C or below until use. Any increase in the temperature of the cryovials before an experiment threatens the viability, functionality, and activity of the hepatocytes.
No. The overlay will interfere with transfection. For transfection assays, we recommend that you perform the transfection first, and then proceed to overlay the cells.